EGFR-targeted fluorescence molecular imaging for intraoperative margin assessment in oral cancer patients: a phase II trial

Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.

shown that systemic administration of these compounds is safe and tumor specific. These findings prompted us to design this innovative application in a clinical trial for the intraoperative assessment of tumor margins during surgical treatment of HNSCC using cetuximab-IRDye800CW. The study is subsidized by the Dutch Cancer Foundation.

Objectives
The main purpose is to establish the intraoperative use of cetuximab-IRDye800CW as a reliable marker for residual tumor in resection margins after surgical removal of HNSCC. The objective is to establish the positive predictive value of cetuximab-IRDye800CW fluorescence as a marker for a tumor positive resection margin.

Study design
The study is designed as a phase 1-2, single center prospective cross sectional diagnostic study in patients with HNSCC that require surgical excision. First, a dose finding study will be performed in 9 patients using 10, 25 and 50 mg of cetuximab-IRDye800CW with three patients per dose cohort. In the first and only performed study at the University of Alabama (UAB) using cetuximab-IRDye800CW in the visualization of HNSCC, the dose found to be optimal was 25mg/m2. We therefore think that a sufficient dose will be found within the proposed range. The most optimal dose from the three studied doses will be used in the second part of the study which will include a cohort of 70 patients. The choice of cetuximab-IRDye800CW dose will be a balance between the lowest dose vs. a clinically usable tumor to background ration (TBR) on the fluorescence images.
During the second phase of the study tumor margins will be studied in a cohort of 70 patients to determine the positive predictive value of optical imaging to identify positive margins.
Based on historical data retrieved from our HNSCC database at UMCG we anticipate in a cohort of 70 patients at least 14 (20%) margin-positive patients and a 90% EGFR overexpression rate. We anticipate a sensitivity of 90% of the cetuximab-IRDye800CW conjugate based on the EGFR overexpression rate, which we will be able to measure with sufficient precision ( 95%CI of 60-96%).

Study population
Patients eligible for inclusion should suffer from a squamous cell carcinoma in the head and neck region (HNSCC) of which the head and neck tumor board of the UMCG has advised to be treated by surgical removal. Detailed description of inclusion and exclusion criteria are given in paragraph 2.3.

Patient related study procedures
Tracer administration: patients will visit the hospital four days prior to the planned surgery of their HNSCC. The cetuximab-IRDye800CW will be injected by slow infusion and patients will be monitored for potential side effects. The dose will be either 10, 25 or 50 mg of cetuximab-IRDye800CW which is less or equal to10% of the dose of cetuximab when used for curative treatment of HNSCC (usually 400mg/m2 loading dose and 250mg/m2 maintenance dose).

Study aims:
The aim is to identify squamous cell carcinoma as fluorescent spots in the margin of a tumor resection specimen or in the wound bed in the patient.
Parameters: Fluorescence imaging and spectroscopy: Fluorescence images will provide an overview of where cetuximab-IRDye800CW fluorescence is located in the resection specimen and the wound bed in the patients. The intra operative camera is very sensitive for cetuximab-IRDye800CW fluorescence. One drawback is that on the fluorescence image the exact depth from which the fluorescence signal is generated cannot be established. Furthermore, most likely there will be background fluorescence signals from normal tissue.
Therefore, confirmation of the fluorescence signal on images requires quantification of the fluorescence signal. This can be performed by using a spectroscopy technique (MDSFR spectroscopy) that can quantify (in M/m 3 ) specifically IRDye800CW-fluorescence by placing a fiber tip in contact with the tissue. This spectroscopy technique has a shallow sampling depth of 1-2 mm. If fluorescence is generated from deeper layers, the signal of spectroscopy will be low (only background signal from muscle, connective tissue and salivary glands). If the IRDye800CW-fluorescence signal is generated from tumor in the resection margin the fluorescence signal will be much higher because SCC-tumor cells overexpress EGFR. The parameter that will be established is the threshold level at which background cetuximab-IRDye800CW spectroscopy signal can be separated from much higher spectroscopy signals of cetuximab-IRDye800CW accumulated in tumor.

Pathology:
The tumor specimen will be processed for histology according to the current standard used in clinical cancer care. Diagnosis on margins, selected histological features necessary for clinical decision making will be provided. Next to this fluorescence images will be collected from the tumor specimen and biopsies. Margin width and number of positive margins will be noted and correlated to the location of fluorescent locations in the margins.
From this positive predictive value will be calculated.

Burden -Time investment:
Patients need to make one extra visit to the UMCG four days before their planned surgery that will take approximately 2 hours. Usually patients are admitted one day prior to the planned surgery. Therefore the measurements one day before surgery will not require extra time investment Burden-extra procedures: 1) Intravenous administration of cetuximab-IRDye800CW.

2)
Fluorescence images will be taken from the tumor one day prior to surgery in the first cohort of nine patients.
3) The estimated time for taking fluorescence images and spectroscopy measurements is approximately 30min. Therefore the time under general anesthesia will be prolonged. The usual time of surgical procedures for removal of HNSCC ranges from 2 hours to 15 hours, depending on complexity of the surgical procedure. 4) from the wound bed in the patient that exists after tumor excision, biopsies will be taken in the ongoing general anesthesia, of spots positive of cetuximab-IRDye800CW as seen on the fluorescence imaging and confirmed by spectroscopy. Risks: Allergic reactions to cetuximab have been reported but this is considered a low risk.
No preclinical or clinical study reported higher than grade 2 adverse events. the first study with cetuximab-IRDye800CW no serious events were reported in six patients.
Benefit: Patients will have no benefit from this study directly. Surgery will be planned as usual. During surgery, no decisions will be made based on the fluorescence imaging. The benefit of this study will be the establishment of usefulness of cetuximab-IRDye800CW during surgery to identify margins containing tumors. The results of these types of study will be at least beneficial for other patients with cancer in the future. Clinical experience will be obtained with fluorescent labeled antibody in intra operative margin assessment during surgery of HNSCC.  When histopathological evaluation shows that a margin is positive, adjuvant treatment is necessary. In cases of small tumors (T1) or a superficially located positive margin a reresection can be performed. However, often (87% of the cases) the margin is positive at a deeper seated margin in the deeper connective tissue layers. Usually it is not possible to exactly locate the region of the positive margin and the choice for adjuvant therapy than will be post-operative concomitant chemoradiation. Therefor a method that will aid the surgeon to identify during surgery where the margin is close could be of great benefit for the patient and reduce the morbidity of the post-operative treatment.

Intraoperative near infrared fluorescence imaging in cancer
Until recently, the application intraoperative near infrared fluorescent imaging was limited due to a lack of specificity of fluorescent probes for tumor tissues.  In recent years, significant progress is made in the development of optical imaging devices for intraoperative near infrared fluorescence (NIRF) imaging. In collaboration with the Technical University of Munich (TUM), a real-time optical imaging system has been developed using video-rate concurrent multi-spectral near infrared and visible light imaging at a molecular level. (20,21) Following the intravenous administration of an fluorescent tracer, an external light source with a defined wavelength is used to illuminate the subject. As light propagates through the tissue, it will excite both surface and subsurface localized optical contrast agents (22). Immediately after excitation, the contrast agent responds by releasing low-energy light of a longer wavelength, which can subsequently be detected by a highly sensitive charge-coupled (CCD) camera. More recently, the company SurgVision improved the initial TUM setup by introducing the SurgVision F2 multispectral imaging system. This updated system is currently in use at the UMCG. Improvements include upgrades of crucial white light elements, white light camera, the CCD and optical filter sets. This resulted in a higher and more efficient signal excitation and therefore better fluorescent signal visualization. The setup was further improved by adding connectors for fiber optic devices, which made a connection with endoscopic devices possible. This allows for a more easy access of anatomically deeper situated areas such as the esophagus, colon and the oral cavity for NIRF imaging. superficial measurements with relatively short photon path-lengths. As a result, the number of scattering events is relatively small. This means that the angle in which the light is scattered becomes increasingly important for the amount of reflected light. This "scattering angle" follows a probability distribution that is described by the (unknown) tissue phase function. Because the reflectance in MDSFR is highly dependent on this phase function it is necessary to characterize it in order to measure the reduced scattering coefficient μs'[mm-1].

In vivo NIRF imaging: Clinical trials performed at the UMCG
The first in-human application of intraoperative tumor-specific fluorescence imaging using  (Table 2). Preliminary data from breast cancer imaging (NCT01508572; NCT02583568) indicate that fluorescence of superficially located tumors can be visualized relatively easy (Figure 1). First results from our trial in patients with familial adenomatous polyps (NCT02113202) show that bevacizumab-800CW NIRF imaging is also feasible using a NIRF endoscopy platform, developed by our collaborators at the Technical University of Munich/Helmholtz ( Figure 2). While the full analysis of the data acquired in the ongoing clinical trials is still underway, the potential of using bevacizumab-800CW for tumor specific molecular imaging is clear.  Table 2 Overview of the ongoing and completed clinical trials using bevacizumab-800CW.

Rationale for the use of cetuximab-IRDye800CW as fluorescent tracer in HNSCC
Multiple agents are currently being applied for targeted therapy in HNSCC. These include bevacizumab, cetuximab and lapatinib. (33) Bevacizumab is a monoclonal antibody that binds to vascular endothelial growth factor (VEGF). A disadvantage of VEGF is that it is overexpressed in both HNSCC and peritumoral vasculature and lymph beds. The higher background signals of these tissues make bevacizumab less attractive for fluorescence imaging. (34) Recently, we imaged solid tumors in animals using bevacizumab and  assessed. Tumors were imaged using a real-time NIRF imaging system (NOVADAQ). The surgical pathology specimens of both tumor and normal tissues were imaged using a closed field imaging system (Odyssey) prior to histological sectioning. These results indicate that the application of cetuximab-IRDye800CW for intraoperative fluorescence imaging is feasible and safe. This prompted us to aim for the clinical translation of cetuximab-IRDye800CW for intraoperative application in HNSCC. The exact demarcation  therefore decided to perform a dose finding study of cetuximab-IRDye800CW in HNSCC in our setting using the SurgVision F2 multispectral imaging system with an attached nasopharyngeal endoscope (see section 7 for detailed information of devices used in this study) to identify the most optimal dose of cetuximab-IRDye800CW for our imaging system.
The dose of 2.5 mg/m 2 as administered in the dose escalation study can be considered microdosing and is comparable to the dose of 4.5 mg used in ongoing UMCG studies using bevacizumab-800CW. From these studies we have learned that a dose of 4.5 mg bevacizumab-IRDye800CW is too low to reliably measure a fluorescence signal or perform adequate spectroscopy (personal communication dr. Nagengast (UMCG) and dr. Robinson (Erasmus Medical Center)). Furthermore, the dose escalation study performed at the UAB reported TBR levelling with higher cetuximab-IRDye800CW doses (See section 1.6). Also because of the higher sensitivity of our system as found by calibrating both devices and the TBR levelling with higher cetuximab-IRDye800CW doses, we hypothesized that a lower maximum dose than administered at the UAB may be sufficient for an adequate TBR in our setting. The administrated doses of cetuximab (10, 25 and 50mg) as proposed for this study are regarded safe (see section 6.4) and correspond to a fraction of the therapeutic dose (400mg/m 2 loading dose and 250mg/m 2 maintenance dose) which is given during concurrent chemoradiation. For these reasons we think it is appropriate to select doses of 10, 25 and 50mg of cetuximab-IRDye800CW and test each dose in a cohort of 3 patients.

Primary objective Part 1 (dose finding)
-To determine the optimal dose of cetuximab-IRDye800CW for intra operative imaging with the SurgVision F2 multispectral imaging system for easily accessible anatomical areas in the head and neck region, or with a standard nasopharyngeal endoscope attached to the SurgVision F2 multispectral imaging system for difficult accessible anatomical areas in the head and neck region.

Primary objective Part 2 (main study)
-The main purpose is to establish the intraoperative use of cetuximab-IRDye800CW as a reliable marker for residual tumor in resection margins after surgical removal of HNSCC. The objective is to establish the positive predictive value of cetuximab-IRDye800CW fluorescence as a marker for a tumor positive resection margin. -To obtain information on safety aspects of cetuximab-IRDye800CW administration by registration of conjugate blood levels, conjugate integrity, side effects, adverse events (AE), serious adverse events (SAE) and suspected unexpected serious adverse reactions (SUSAR).

End points Part 1 (dose finding)
The primary endpoint of the dose finding study is to identify a dose corresponding to a sufficient Tumor to Background Ratio (TBR). The TBR will be calculated by a combined assessment of intraoperative in vivo and ex vivo fluorescent signals (SurgVision F2 multispectral imaging system, nasopharyngeal endoscope, MDSFR/SFF spectroscopy) together with ex vivo examinations (histological examination, NIR flatbed scanning and fluorescence microscopy).
Secondary endpoints will be: 1) Safety data (cetuximab-IRDye800CW blood levels and conjugate integrity, side effects, AE, SAE, SUSAR); 2) Localizing patterns of cetuximab-IRDye800CW fluorescence within both tumor and normal tissue by fluorescence microscopy and histological examination.

End points Part 2 (main study)
The

STUDY DESIGN
The current study is a non-randomized, non-blinded, prospective, single center, crosssectional diagnostic study. A maximum of 88 patients with HNSCC will be included. All The study consists of two parts. In part 1, the optimal cetuximab-IRDye800CW for intraoperative imaging will be determined. In part 2, determine the threshold level of cetuximab-IRDye800CW fluorescence for reliable intraoperative deep margins assessment with high sensitivity while ensuring an adequate positive predictive value will be determined.
A detailed description of the rationale of the imaging protocol can be found in section 3.3.

Study design Part 1 (dose finding)
Part 1 of the study will consist of 3 study cohorts with a maximum of 3 patients for each cohort. The doses for each consecutive cohort will increase from 10, 25 to ultimately 50 mg setting. Depending on the outcome of the DSMB review in terms of (S-)AEs we will continue with part 2 of our study. If necessary, the DSMB may advise to increase each dose cohort to 6 patients (3+3), thereby increasing the maximum number of patients included in part 1 of the study to 18 ( Figure 5).

Study design part 2
Part 2 of this study will consist of 70 patients. The administered dose is based on the optimal cetuximab-IRDye800CW dose as identified in part 1 of our study during the dose finding. The rationale of this sample size calculation can be found in section 4.4 "Sample size calculation." An interim analysis will be performed following the inclusion of 35 patients.
Depending on the outcome of the DSMB review in terms of (S-)AEs we will resume with inclusion of the final cohort of 35 patients ( Figure 5). 12) Magnesium, potassium and calcium lower than the lower limit of normal range.

Sample size calculation
Patients will be selected from the group of patients that will be surgically treated for HNSCC at the UMCG. The maximum number of patients included for this study will be 88 (Part 1: 9(+9) and Part 2: 70) if the DSMB advises to include more patients following the interim analysis in the dose finding study. The patients will be included within 4 years. We are confident that it is reasonable to include the required amount of patients within in the Head and Neck unit of the UMCG during this time frame. In 2015, more than 100 operations for removal of HNSCC were performed at the department of Oral and Maxillofacial Surgery of the UMCG.

Sample size calculation Part 1 (dose finding)
The primary aim of part 1 is to identify the optimal dose of cetuximab-IRDye800CW for intraoperative detection of HNSCC, defined as a sufficient TBR. The dose finding part involves a small sample size of 9 patients (3 cohorts with 3 patients each). Based on prior results from the UAB, we estimate that this sample size is sufficient to verify an optimal dose in our setting. An interim analysis will be performed following the inclusion of all 9 patients.
This interim analysis will be discussed with the DSMB. If data is insufficient, a maximum of 9 additional patients will be included for analysis in the dose finding study.

Sample size calculation Part 2 (main study)
Part 2 will include 70 patients. We expect that 20% (n=14) of the included patients will have positive deep margins. As each patient may have more than one ROI with positive margins, and as fluorescence positive ROIs will be sampled already at a low signal, this will lead to sufficient data to evaluate the spectroscopy signal for discriminative value and derive an optimal threshold (given the necessary 10 events per predictor). Furthermore, we will use bootstrapping methods to correct for optimism in threshold finding. With regard to the perpatient analysis, we expect that approximately 90% of patients with a positive margin will show EGFR overexpression in that margin and will thus be detected by the spectroscopy signal. If true, this will lead to 12 of 14 patients with positive margins to be detected. Given the sample size, this results in a sensitivity of 86% with a corresponding 95% C.I. of 60-96% (at 71% sensitivity, the 95% C.I. will be 45-88%, and at 50%: 27-73%), yielding sufficient precision as to the expected impact on margin status of imaging-guided surgery and thus allowing the informed design of a subsequent comparative randomized study. Estimates of specificity will be even more precise given the larger number of patients with negative margins.

Investigational product/treatment
The investigational product is cetuximab-IRDye800CW. This is in the literature also sometimes referred to as cetuximab-800CW or cetuximab-IRDye800. In this protocol it is also referred to as 'the tracer' or 'conjugate'. Cetuximab-IRDye800CW is administered intravenously 4 days before routinely scheduled surgery at multiple increasing doses (10,25 and 50 mg) during part 1 of this study. The optimal dose of cetuximab-IRDye800CW as identified in part 1 of this study is administered intravenously 4 days before routinely scheduled surgery in 70 patients during part 2 of this study.

Name and description of investigational product(s)
The

Description and justification of route of administration and dosage
The tracer will be administered intravenously. This route of administration equals the

Dosages, dosage modifications and method of administration
The tracer is administered by intravenous infusion. Cetuximab-IRDye800CW is provided as a ready to use solution (1mg/1ml). Preparation of a dose for use in the study consists of drawing the required volume of tracer into a syringe of sufficient size. No dilution steps will be performed on the product. Patient doses of 10, 25 or 50mg will be administered, therefore a syringe containing 10, 25 or 50ml will be prepared.

Preparation and labeling of Investigational Medicinal Product
All conjugates used in this study will be prepared, packaged and labeled under the

Drug accountability
There are no special requirements for the shipment, receipt, disposition, return and destruction of cetuximab-IRDye800CW.

Name and description of non-investigational product(s)
The intraoperative imaging of cetuximab-IRDye800CW will be performed using a

SurgVision F2 multispectral imaging system
The multispectral F2 camera system provided by SurgVision is used for intraoperative NIRF compared to ensure that study outcomes will not be biased by major differences in equipment used. It was found that the UMCG used system is much more sensitive in detecting fluorescence than the system used at UAB (see attachment K6.6). Even when a standard nasopharyngeal endoscope was coupled to the SurgVision camera, this set-up outperformed the system used at UAB. Since the outcome measurements will be the TBR, which is a relative measure, the difference between the "open air" setting of the SurgVision multispectral imaging system and the nasopharyngeal endoscope will not influence the outcome of the study. The imaging system will be used to visualize the tracer and to guide the positioning of the MDSFR/SFF spectroscopy fibre. The MDSFR/SFF spectroscope will perform quantitative measurements at the fluorescent spots that will proved the data for this study to calculate the positive predictive value.

Flexible Nasopharyngeal endoscope
The "open air" SurgVision multispectral imaging system might not be able to image areas in the oral cavity or oropharynx due to anatomical restrictions. A standard flexible nasopharyngeal endoscope will be coupled to the SurgVision system to visualize the tumor and surgical wound bed following excision in these areas. Additional information about specifications and quality assurance of the nasopharyngeal endoscope is provided separately (attachment K6.3.1 and K6.3.2 respectively). The nasopharyngeal endoscope that will be used is a standard endoscope which is routinely used in the UMCG for inspection of the upper aerodigestive tract. When coupled to the imaging system it is possible to gain fluorescence images of sufficient quality and sensitivity. In attachment K6.6 the sensitivity of the SurgVison camera (open air or with the nasopharyngeal endoscope attached) compared to the system used at UAB for the first human trial of cetuximab-IRDye800CW. Even the endoscope the UMCG system is much more sensitive than the fluorescence imaging system from UAB.

Summary of known and potential risks and benefits
The equipment that is to be used in this study is certified by drs. F. Boorsma, head of the department of medical technology of the UMCG and qualified specialist concerning Sterile Medical Devices (DSMH) and matters relating Scope Cleaning and Disinfection (DSRD) (see attachment K6.8.1 and 6.8.2). A FMEA risk analysis has been performed for the SurgVision F2 multispectral imaging system and MDSFR/SFF probe (see attachments K6.2 (SurgVision F2 multispectral imaging system) and K6.5 and K.6.6 (MDSFR/SFF probes)).

Preparation and labelling of Non Investigational Medicinal Product
Not applicable

Drug accountability
Not applicable

Other study parameters (if applicable)
Patient characteristics (age, sex, BMI, history and morbidity, localization and classification of cancer, treatment outcome, blood pressure, pulse and temperature before and after tracer administration, baseline blood count/ liver and kidney function, signs and symptoms before and after tracer administration.
Histopathologic examinations related to ex vivo EGFR expression and cetuximab-IRDyeCW800 distribution.

Randomization, blinding and treatment allocation
The current study is a non-randomized, non-blinded single center study, in which all patients undergo the same surgical procedure according to standard care. For part I of this study there will be a difference in administered tracer dose; the first 9 patients will be in the 3 Here, there's no randomization, blinding or treatment allocation applicable since this is a prospective cross sectional diagnostic study.

Study procedures
General clinical practice will have priority over study procedures at all times. The patients will be informed about the aims of the study, the possible adverse events, the procedures and possible hazards to which they will be exposed before enrolment into the study. They will be informed as to the strict confidentiality of their patient data.
Patients will be contacted by phone, mail or e-mail to ask if they are interested in participating in this study by one of the investigators (treating/research physician) involved in this study.
A flow chart from part 1 and part 2 of the study is provided in Figure 6. For more specified information about informed consent, see chapter 11.2.

Visit 1:
Tracer administration and safety monitoring will occur at the day of administration of the tracer, the patient is asked for signs/symptoms present.
Before administration of the tracer, a pretreatment ECG is performed and vital signs will be measured. A QT prolongation of >440 ms in males or >450 ms in females is not to be accepted and patients will be excluded from further study participation. An intravenous line will be installed. Blood will be drawn during the pre-operative screening, will be used as baseline parameters in case of any adverse events after administration. A blood sample will be taken prior to infusion of cetuximab-IRDye800CW to determine baseline plasma concentration (T=0) (only in dose-escalating cohort). A single dose of 10-25-50 (depending on cohort) will be administered intravenously to the patient. The infusion line will be flushed with a saline solution afterwards. After tracer administration, blood pressure, pulse and temperature will be measured. A second blood sample will be taken to determine plasma concentration and tracer stability one hour after administration (T=1) (only in dose-escalating cohort). All above will be registered by filling in the CRF. The patient will be observed for 60 minutes following tracer injection. During safety monitoring, a crash car with necessary equipment is available in case of an adverse reaction. All above procedures will be performed by the research physician.

Visit 2
One day prior to surgery The patient will be hospitalized, as standard surgery protocol requires, and is asked for signs/symptoms that occurred since the tracer administration or the last visit. Vital signs will be measured. Fluorescence images and MDSRF spectra will be acquired of tumor and normal tissue around the tumor region (only in dose-escalating cohort), above will be performed by research physician. A third blood sample will be taken to determine plasma concentration and tracer stability (T=2) (only in dose-escalating cohort).

Day of surgery
The patient is asked for signs/symptoms that occurred since the tracer administration or the last visit. The patient will be brought to narcosis following the standard protocol within the UMCG. A fourth blood sample will be taken to determine plasma concentration and tracer stability (T=3) (only in dose-escalating cohort). A technical assistant will be present to operate the SurgVision multispectral camera and to assure relevant images are saved in close communication with the surgeon (treating physician). Fluorescence images and MDSRF spectra will be acquired of tumor and normal tissue around the tumor region, and the tumor will be excised following the standard protocol. After tumor removal, the woundbed will be inspected and again fluorescence images and MDSRF spectra will be applied.
Fluorescent areas in the woundbed will be analyzed by MDSFR spectroscopy and regions of interest will be biopsied. There will be no re-resection as a result of the new obtained biopsy data. Subsequently these biopsies will be processed separately for histopathology and fluorescence microscopy.. Decisions on post-operative strategies will otherwise not be influenced by this study.
After the operation patient will go to the nursing ward and will be strictly observed following the UMCG post-operative protocol. On the first and second day post-operative, blood samples will be drawn to determine plasma concentration and tracer stability (T=4 and T=5) (only in dose-escalating cohort).

Specimen related study protocol
After completion of the resection, the resected specimen will be analyzed immediately after removal by using ex vivo fluorescence imaging and MDSFR spectroscopy. On the excised tumor specimen we will acquire ex vivo fluorescence images and perform MDSFR-point spectroscopy of carefully selected regions of fluorescence. The regions that display cetuximab-IRDye800CW-fluorescence will be inked with non-fluorescent ink after ex vivo fluorescence imaging. These marked areas will be identified by the pathologist for histopathology analysis for the presence of tumor in the subsequent fluorescence microscopy. Important to state, the specimen processing of the excised tumor will be done with no interference with standard of care. The excised tumor specimen and the separately taken biopsies from the surgical woundbed will be submitted for standard HE histopathology, for assessing the margin status and other descriptive data necessary for clinical use. In the unexpected case that the study-based separately sampled biopsies from the surgical woundbed show tumor, this finding will be used for the decision by the Head and Neck tumor board on post-operative strategy.
Decisions on post-operative strategies will otherwise not be influenced by this study. Next to assessment of the margin status by routine histopathology, the inked areas will be specifically analyzed for margin status. Diagnosis of the separately taken biopsies from the

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject due to failure of tracer administration or urgent medical reasons. Direct withdrawal should be considered in case of a serious adverse event.

Specific criteria for withdrawal (if applicable)
Not applicable.

Replacement of individual subjects after withdrawal
Patient who are withdrawn will be replaced in this study.

Follow-up of subjects withdrawn from treatment
After administration of the tracer, (serious) adverse events that occur in subjects that are withdrawn from the study procedure, will still be recorded if possible.

Termination based on safety aspects
A multidisciplinary team with study investigators will discuss safety aspects during the study procedures. Results will be reported immediately in case of any SUSAR to the external Data Safety Monitoring Board (DSMB). After the first 9 patients of part 1 and whenever (serious) adverse events ((S)AE) happen, these will be reported to the DSMB. The study will be considered to be terminated in case a suspected unexpected serious adverse reaction (SUSAR) occurs in any of the patients, according to advice of the DSMB.

Termination based on Cetuximab-IRDye800CW accumulation
This study will be suspended immediately if any serious adverse event related to the administration of the tracer occurs in any of the patients. The design of this study warrants maximal data collection, while risks and burden for patients are minimized. Also, the framework of this study can be used for evaluation of other, newly developed (fluorescent) tracers. The study will not be terminated if after interim analysis of the first 9 evaluable patients no uptake of Cetuximab-IRDye800CW in SCC tissue can be shown by any of the available technologies; if this occurs the study will continue with inclusion of patients in a new cohort of dose finding

Termination based on other aspects
The study will be suspended based on urgent medical or ethical considerations as decided by the principal investigators. In case of termination of the study, the institution, regulatory authorities, CCMO and the METC of the study center will be informed.

Temporary halt for reasons of subject safety
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if there is sufficient ground that continuation of the study will jeopardize subject health or safety. The sponsor will notify the accredited METC without undue delay of a temporary halt including the reason for such an action. The study will be suspended pending a further positive decision by the accredited METC. The investigator will take care that all subjects are kept informed.

Adverse events (AEs)
Adverse events are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to [the investigational product / trial procedure/ the experimental intervention]. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.

Serious adverse events (SAEs)
A serious adverse event is any untoward medical occurrence or effect that results in death; is life threatening (at the time of the event); requires hospitalization or prolongation of existing inpatients' hospitalization; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; or any other important medical event that did not result in any of the outcomes listed above due to medical or surgical intervention but could have been based upon appropriate judgement by the investigator.
An elective hospital admission will not be considered as a serious adverse event.
The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited METC that approved the protocol, within 7 days of first knowledge for SAEs that result in death or are life threatening followed by a period of maximum of 8 days to complete the initial preliminary report. All other SAEs will be reported within a period of maximum 15 days after the sponsor has first knowledge of the serious adverse events.

Suspected unexpected serious adverse reactions (SUSARs)
Adverse reactions are all untoward and unintended responses to an investigational product related to any dose administered. Unexpected adverse reactions are SUSARs if the following three conditions are met: 1.
The event must be serious (see chapter 9.2.2);

2.
There must be a certain degree of probability that the event is a harmful and an undesirable reaction to the medicinal product under investigation, regardless of the administered dose; 3. the adverse reaction must be unexpected, that is to say, the nature and severity of the adverse reaction are not in agreement with the product information as recorded in: -

Summary of Product Characteristics (SPC) for an authorized medicinal product;
-Investigator's Brochure for an unauthorized medicinal product.
The sponsor will report expedited the following SUSARs through the web portal The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted once every half year to the METC. This line-listing provides an overview of all SUSARs from the study medicine, accompanied by a brief report highlighting the main points of concern.
The expedited reporting of SUSARs through the web portal Eudravigilance or ToetsingOnline is sufficient as notification to the competent authority. The sponsor will report expedited all SUSARs to the competent authorities in other Member States, according to the requirements of the Member States.
The expedited reporting will occur not later than 15 days after the sponsor has first knowledge of the adverse reactions. For fatal or life threatening cases the term will be maximal 7 days for a preliminary report with another 8 days for completion of the report.

Annual safety report
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year throughout the clinical trial, a safety report to the accredited METC, competent authority, and competent authorities of the concerned Member States.
This safety report consists of: a list of all suspected (unexpected or expected) serious adverse reactions, along with an aggregated summary table of all reported serious adverse reactions, ordered by organ system, per study; a report concerning the safety of the subjects, consisting of a complete safety analysis and an evaluation of the balance between the efficacy and the harmfulness of the medicine under investigation.

Follow-up of adverse events
All AEs will be followed until they have abated, or until a stable situation has been reached.
Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.
SAEs need to be reported till end of study within the Netherlands, as defined in the protocol

Data Safety Monitoring Board (DSMB)
An independent external Data Safety Monitoring Board of experts is established to perform ongoing safety surveillance and to evaluate interim analyses on the safety data. This board is necessary considering the available knowledge about Cetuximab-IRDye800CW, see chapter 13.
The independent members of the DSMB are: The investigators involved in this study will perform an interim analysis on safety data, after the first cohort of 9 patients. Interim analyses are performed according to chapter 10.4. The investigators will report to the independent DSMB. The DSMB will consider essential parts of study conducts like protocol adherence, patient withdrawal and safety. The DSMB will work according to Standard Operating Procedures (SOPs). A DSMB charter is available.
The responsibilities of the DSMB include: -Decide whether to recommend that the trial continues to recruit participants or whether recruitment should be terminated either for everyone or for some participant subgroups.
The DSMB will discuss the results of the interim-analysis and advice the steering committee.
The DSMB provides recommendations regarding study modification, continuation or termination. Discontinuation of the trial is advised by the DSMB according to the pre-defined stopping guidelines stated in paragraph 8.7. The advice(s) of the DSMB will only be sent to the sponsor of the study. Should the sponsor decide not to fully implement the advice of the DSMB, the sponsor will send the advice to the reviewing METC, including a note to substantiate why (part of) the advice of the DSMB will not be followed.

Descriptive statistics
Patient descriptive data will include TNM status, tumour size, localization, margin status.
Only during the dose finding study, integrity of cetuximab-IRDye800CW will be monitored and described.

Study parameters
The primary aim of part 1 is to determine whether the average TBR as observed in 9 patients is sufficient. The primary aim of part 2 of the study is to define a threshold value for fluorescence imaging-guided spectroscopy signal that allows the detection of tumour precision-recall curve from which we will derive the optimal threshold value. As each patient may contribute multiple ROIs -thereby increasing statistical power -, we will take clustering into account by multilevel analysis. Furthermore, we will include bootstrapping methods for the threshold finding to correct for optimism (i.e. to ensure that the proposed threshold will not only hold true in the data it derived from, but also in new patients).
Next, we will evaluate the defined spectroscopy threshold value on a per-patient level (true

Interim analysis
An interim analysis towards the primary endpoint will be conducted by the investigators involved in this study after the first 9 patients (3 patients per dosage) of the dose finding study. If no adequate tumor-to-background ratios are observed in the first 9 patients with HNSCC, the dose finding study might be extended.
An interim analysis towards the key safety parameters including cetuximab-IRDye800CW induced AE's, SAE's and SUSARS will be performed after the first 9 patients (see also the DSMB charter and sections 8.7.2 and 9.5 of this protocol). The results will be reviewed by the external independent data monitoring committee (DSMB).

Regulation statement
The study will be conducted according to the principles of the Declaration of Helsinki (Fortaleza, Brasil, 2013 amendment) and in accordance with the medical Research Involving Human Subjects Act (WMO) and other guidelines, regulations and Acts. The protocol has been written and the study will be conducted according to the ICH Harmonized Tripartite Guideline for Good Clinical Practice. The protocol will be approved by the Local, Regional or National Ethics Committees. with the standard information each patient receives prior before the scheduled operation (see patient information leaflets for UMC Groningen).

Recruitment and consent
. The patients will be informed about the aims of the study, the possible adverse events, the procedures and possible hazards to which they will be exposed before enrolment into the study. They will be informed as to the strict confidentiality of their patient data.
Patients will be contacted by phone, mail or e-mail to ask if they are interested in participating in this study by one of the investigators (treating/research physician) involved in this study.
Each patient will be given the opportunity to ask questions and will be informed about the right to withdraw from the study at any time without prejudice. See the patient information sheet and patient informed consent statement for the UMC Groningen.
When patients intend to participate, the visit for tracer administration with a pretreatment ECG is planned and patients will be asked to bring the signed informed consent form, which will be received by the research physician.

Informed consent
Documented informed consent must be obtained for all patients included in the study before they are registered in the study. Patients must be given adequate opportunity to read the information and enquire about details of the study before consent is given. The informed consent procedure takes place conform the ICH guidelines on Good Clinical Practice. This implies that the written informed consent form will be signed and personally dated by the patient or by the patient's legally acceptable representative. The informed consent statement will be signed and dated by the research physician afterwards and the patient will receive a copy. The general physician of each patient will be informed about the enrolment of the patient to the study.

Objection by minors or incapacitated subjects (if applicable)
Not applicable

Benefits and risks assessment, group relatedness
For the participating patients, there is no diagnostic or treatment benefit related to the study.
Participation may possibly produce useful scientific data for the future. Risks related to the imaging procedure and spectroscopy procedure is very minimal, as shown in the preliminary data (UAB). The surgical planning will not be altered due to the fluorescence imaging during this study. During surgery, delineation of margins will not be influenced by the intraoperative imaging data. The fluorescence imaging will not influence surgical decisions. The type of surgery will not be influenced by the study protocol.

Compensation for injury
See attachment G1 and G2 Briefly, the sponsor/investigator has a liability insurance which is in accordance with article 7 of the WMO.
The sponsor (also) has an insurance which is in accordance with the legal requirements in the Netherlands (Article 7 WMO). This insurance provides cover for damage to research subjects through injury or death caused by the study. The insurance applies to the damage that becomes apparent during the study or within 4 years after the end of the study.

Incentives (if applicable)
For each day of patient related study procedures, the subjects will receive compensation for travelling expenses (€ 0.19/km) and a ticket for free parking.

Case Record Forms
Case Record Forms (CRFs) will be provided by investigator. The CRFs will be completed by the investigators. The investigators are responsible for the legibility, completeness and correctness of the CRF. The signature of the investigator at the end of each chapter of the CRF will serve as a confirmation. Errors, changes and/or additions entered on original CRFs must be corrected by drawing in a single line through the incorrect entry and writing the new entry as close to the original as possible as so to leave the correct entry legible. If necessary the reason for the change must be given. The correction must be initialed and dated by the authorized person making the change.

Data storage
Data of patients will be handled confidentially and a coded identification number (study protocol number 'ICON' followed by number of inclusion: 01) will be used to link the data to the specific patients. The data that can be linked to a specific patient will be stored separately. The medical investigator safeguards the key to the code. The handling of the personal data complies with the Dutch Personal Data Protection Act (in Dutch: de Wet bescherming persoonsgegevens). These data will be stored at the specific site for at least twenty years. Coded / anonymized study data will be made available to our relevant partners within the project; Erasmus Medical Center, Rotterdam and University Medical Center Utrecht. They will assist in the use and maintenance of the MDSRF spectroscope. Moreover, the will perform parts of the data analysis of the MDSRF spectroscope.

Monitoring and Quality Assurance
On-site monitoring will take place conform the NFU (Nederlandse Federatie van Universitair Medische Centra)-guideline "Kwaliteitsborging van mensgebonden onderzoek 2010" by the appointed monitor. For this study, the risk classification is considered "high", which implies intensive independent monitoring of at least 3 visits per year, dependent on the patient inclusion speed. This study will be monitored by an independent, certified monitor, employed by the UMC Groningen The monitor will perform source data verification on the research data by comparing the data entered into the CRF with the available source documentation and other available documents. Source documents are defined as the patient's hospital medical records, clinician notes, laboratory print outs, digital and hard copies of imaging, memos, electronic data etc.

Data Verification
The monitor will verify the following items (100%): Patient flow (inclusion speed and dropout rate); Informed consent forms (presence, dates, signatures); Trial Master File and laboratories, pharmacies and other departments will be assessed

Amendments
Amendments are changes made to the research after a favorable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favorable opinion.
A 'substantial amendment' is defined as an amendment to the terms of the METC application, or to the protocol or any other supporting documentation, that is likely to affect to a significant degree: the safety or physical or mental integrity of the subjects of the trial; the scientific value of the trial; the conduct or management of the trial; or the quality or safety of any intervention used in the trial.
All substantial amendments will be notified to the METC and to the competent authority. Nonsubstantial amendments will not be notified to the accredited METC and the competent authority, but will be recorded and filed by the sponsor.

Annual progress report
The sponsor/investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments.

Temporary halt and (prematurely) end of study report
The sponsor will notify the accredited METC and the competent authority of the end of the study within a period of 90 days. The end of the study is defined as the last patient's last visit.
The sponsor will notify the METC immediately of a temporary halt of the study, including the reason of such an action. In case the study is ended prematurely, the sponsor will notify the accredited METC and the competent authority within 15 days, including the reasons for the

Potential issues of concern
a. Level of knowledge about mechanism of action Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types and differentially expressed in normal tissue versus HNSCC.
Cetuximab is used extensively in the clinic for its anti-neoplastic properties.
IRDye800CW is designed for antibody, protein, or peptide labeling. IRDye800CW has excitation/emission maxima at 774 nm/789 nm, precisely centered in the region known to give optimal signal-to-noise ratio for optical imaging. It is extensively used in preclinical optical imaging experiments for the tracking of probes, since near-infrared fluorophores minimize the optical challenges of detecting photons in tissues.
b. Previous exposure of human beings with the test product(s) and/or products with a similar biological mechanism IRDye800CW solely has never been administered to humans, except in combination with bevacizumab-800CW and cetuximab-IRDye800CW. In animals, the administration of 5 mg/kg of IRDye800CW intravenously showed a fairly consistent peak plasma concentrations.
Toxicity data obtained with IRDye 800CW was published by Marshall et al. (40). Animal toxicological studies on cetuximab-IRDye800CW and preclinical tracer evaluation data showed no adverse effects (45).
More recently, a toxicity study on cetuximab-IRDye800CW has been performed in macaques Details on the nature and seriousness of potential adverse effects are described in 'summary of product characteristics' of cetuximab (see attachment D1, section 4.8, page 7-9).
The initial dose for patients with metastatic carcinoma of the head and neck in combination with radiation or chemotherapy is 450 mg/m 2 body, followed by 250 mg/m 2 once a week.
However, we start our dose finding study at a level of only 10 mg, as we aim to attain a dose as low as possible, which is still sufficient for imaging purposes and safe for the participants.
Not that 10 mg is the absolute dose, and not per kilogram bodyweight.
Potential side effects at a dose on 10-50 mg cetuximab can be summarized as skin rash, hypersensitivity reactions and hypomagnesemia. In the study performed at UAB only grade 1 toxicities occurred. (46) f. Pharmacokinetic considerations Binding to IRDye800cw does not alter the binding capacity of cetuximab, pharmacokinetics or bio distribution, as previously shown. (45) g. Study population The study population are patients with a primary or recurrent HNSCC who will be treated by imaging (38). Based on the preliminary results of our collaborators (UAB), we expect the tracer cetuximab-IRDye800CW to accumulate in EGFR expressing tumors. We will receive direct feedback the moment we visualize the fluorescence in the NIR spectrum using the intra-operative camera. The main advantage of using IRDye800CW for in vivo optical imaging, is the fact that this dye is fluorescent in the near infrared (NIR) spectrum, a spectrum with minimal autofluorescence. Most endogenous fluorophores, in particular hemoglobin, strongly absorb light in the visible light spectrum. Other biological components such as water and lipids strongly absorb light in the infrared region. The combined absorption wavelength of these components translates into a window from approximately 650 to 900nm, near to the infrared light, where the absorption coefficient, and thus autofluorescence, of tissue is at a minimum. Due to this low autofluorescence, the near infrared spectrum (700-900nm) is the optimal window for in vivo, perioperative, HNSCC imaging.
j. Can effects be managed?
A pretreatment ECG will be performed to assure no QTc prolongation is present. Right before cetuximab-IRDye800CW is administered intravenously, blood pressure, pulse and temperature will be measured. There is extensive experience with the administration of cetuximab and the risk of adverse reactions is very small, therefore we decided to observe the patients in this study 60 minutes following tracer injection. Vital signs will be measured after 15, 30, 45 and 60 minutes. During safety monitoring, a crash car with necessary equipment is available in case of an adverse reaction. After the observational period of 1 hour, blood will be drawn, afterwards the intravenous line will be removed and the patient will be discharged with adequate advice to contact the Oral and Maxillofacial surgeon on call if necessary.

Synthesis
Animal toxicological studies on cetuximab-IRDye800CW and preclinical tracer evaluation data showed no adverse effects. (45) A recent clinical study with 89zr-cetuximab showed that only known adverse events to cetuximab were observed, such as skin toxicity, hypomagnesaemia and infusion related reactions, none exceeding grade 2.(47) Cetuximab-IRDye800CW has administered intravenously to 9 patients in the earlier mentioned dosefinding study, no toxicity of the tracer bevacizumab-800IRDyeCW was observed in any of the patients. (39) Up until now, no SAE's or AE's have been reported. Adverse Events may be expected after administration, based on our experience with administrating a much higher dose of unlabeled cetuximab. Hypersensitivity reactions to cetuximab can occur within a short term after administration (up until 1 hour).
Also, hypo-or hypertension can occur after cetuximab administration. However, the expected adverse events are temporal without clinical consequences. This is why the patients will be monitored for one hour after tracer injection, with measurements of vital signs on regular base.
The study protocol with not interfere with general clinical practice. Therefore, surgical risks of patients included in the study protocol will be comparable to patients undergoing a surgical procedure for HNSCC. If necessary, biopsies will be taken after tumor removal. This has in